Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3351. Adviser: Philip M. Best. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2788. Adviser: Milan K. Bagchi. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-02, Section: B, page: 0813. Adviser: Robert V. Farese, Jr.

Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-04, Section: B, page: 2095. Adviser: Robert V. Farese, Jr.

Skeletal muscle is dynamic and actively regenerates following damage or altered functional demand. Regeneration is essential for the maintenance of muscle mass and, when dysregulated as a result of disease or aging, can lead to losses in functional capacity and increased mortality. Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. Therefore, the overall objective of this dissertation was to characterize early molecular alterations in human skeletal muscle to strenuous exercise known to induce a muscle regenerative response. Thirty-five subjects completed 100 eccentric (muscle lengthening) contractions (EC) of the knee extensors with one leg and muscle biopsies were taken from both legs 3 h post-EC. The sample from the non-EC leg served as the control. A well-powered transcriptomic screen and network analysis using Ingenuity Pathway software was first conducted on mRNA from the biopsy samples. Network analysis identified the transcription factor NF-kappaB (NF-kB) as a key molecular element affected by EC. Conformational qRT-PCR confirmed alterations in genes associated with NF-kappaB. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples showed a 1.6 fold increase in NF-kB DNA binding activity following EC. Immunohistochemical experiments then localized the majority of NF-kB positive nuclei to cells in the interstitium, which stained positive for markers of pericyte cells and not satellite cells. To ascertain the mechanistic significance of NF-kB activation following muscle damage, in vitro analyses were carried out using a novel primary pericyte/myoblast co-culture model. Primary pericyte/myoblast co-culture experiments demonstrated that pericytes, transfected with a DNA vector designed to drive NF-kB activation, enhanced proliferation and inhibited myogenic differentiation of co-cultured skeletal muscle myoblasts. Furthermore, reduced NF-kB activation led to enhanced myogenic potential of primary pericytes. Taken together, the data in this dissertation suggest that NF-kB dependent signaling in pericytes regulates myogenic differentiation in a cell- and non-cell autonomous manner and may affect the early regenerative response following muscle damage by inhibiting differentiation and promoting proliferation of muscle satellite cells.

Given the indispensable role of Ca2+ release in fertilization, the type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a key regulatory molecule in mediating the cross-talk between cell cycle progress and the Ca2+ release machinery in mammalian oocytes. The studies in this thesis addressed several important regulatory aspects of the Ca 2+ release activity at fertilization of mammalian eggs. In chapter I, we found that compromised functionality of IP3R1 underlay the defective IP3R1-mediated Ca2+ release in aging eggs. Further, our studies also indicated that modifications on the biochemical status of IP3R1 by incubation with caffeine have positive effects on the Ca2+ release activity and developmental fate of aging mouse eggs. Thus, these results may help facilitate the invention of a cure that could delay/reverse many of age induced detrimental changes thereby restoring the fertilizability of aged eggs. For the next two chapters we mainly focus on the regulatory role of IP3R1 as a significant participant in the early developmental event. Our completed results with the caspase-3 cleaved IP3R1 indicated that the truncated IP3R1 might not play a prominent role affecting the Ca2+ homeostasis during the aging process in mouse oocytes due to its rapid turnover rate. Regardless, we confirmed the leaky property of C-IP3R1 in mouse oocytes and found a novel protective proteolysis pathway in mouse oocytes. Finally development of a system by which we could selectively overexpress IP3R1 phosphorylation mutants allows us to probe important phosphorylation regulatory mechanisms underlying the [Ca2+]i oscillations in mammalian oocytes. Thus the experiments I have done provide valuable information regarding the extensive regulation of the PI pathway, regulation that in aged and non- aged MII eggs, results in a Ca2+ release program that is replete with information.

<p> Many neurological diseases associated with cortical and hippocampal dysfunctions are sexually dimorphic in incidence and have been linked to defects in mRNA splicing. My thesis investigated the sex- and age-related changes in gene expression of six RNA processing genes within the cortex/hippocampus of male and female mice during early development. Gene expression was measured using RT-qPCR with mouse cortex/hippocampus samples collected on the day of birth and one week after birth. Immunoblotting was also used to measure the protein levels of one RNA processing gene to determine whether the expression paralleled mRNA expression. <i>Dhx8 mRNA</i> and <i>Sf3a2 </i> protein expression was found to be sexually dimorphic, while <i> Thoc3, Tut1,</i> and <i>Sf3a2</i> showed mRNA expression changes with age. Sexually dimorphic and age-related expression of these genes suggests that RNA processing may play an important role in brain sexual differentiation and early neural development; however the underlying mechanisms remain to be elucidated.</p>

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<p> Heterotrimeric guanine nucleotide-binding proteins (G-proteins) consist of an alpha subunit (G&agr;) and the dimeric beta-gamma subunit (G&beta;&gamma;). The first example of direct cell signaling by G&beta;&gamma; was the discovery of its role in activating G-protein regulated inwardly rectifying K⁺ (GIRK) channels which underlie the acetylcholine-induced K⁺ current responsible for vagal inhibition of heart rate. Published crystal structures have provided important insights into the structures of the G-protein subunits and GIRK channels separately, but co-crystals of the channel and G&beta;&gamma; together remain elusive and no specific reciprocal residue interactions between the two proteins are currently known. </p><p> Given the absence of direct structural evidence, we attempted to identify these functionally important channel-G&beta;&gamma; interactions using a computational approach. We developed a multistage computational docking algorithm that combines several known methods in protein-protein docking. Application of the docking protocol to previously published structures of G&beta;&gamma; and GIRK1 homomeric channels produced a clear signal of a favored binding mode. Analysis of this binding mode suggested a mechanism by which G&beta;&gamma; promotes the open state of the channel. The channel-G&beta;&gamma; interactions predicted by the model <i>in silico</i> could be disrupted <i>in vitro </i> by mutation of one protein and rescued by additional mutation of reciprocal residues in the other protein. These interactions were found to extend to agonist induced activation of the channels as well as to activation of the native heteromeric channels. </p><p> Currently, the structural mechanism by which G&beta;&gamma; regulates the functional conformations of GIRK channels or of any of its membrane-associated effector proteins is not known. This work shows the first evidence for specific reciprocal interactions between G&beta;&gamma; and a GIRK channel and places these interactions in the context of a general model of intracellular regulation of GIRK gating.</p>

<p> To maintain human health, the lymphatic system requires a structurally and functionally sound network of lymph vessels to absorb lipid-based nutrients, preserve extracellular fluid homeostasis, and mediate immune responses. Aside from lymphedema, investigations in the past few decades have found that impairment of the lymphatic vasculature is also involved in processes such as inflammation, tumor metastasis, fat metabolism, and obesity. However, despite a long history of study and rekindled vigor in the field of lymphatic vascular research, our knowledge of lymph vessel development and physiology is still quite limited. Recently, mutations in a protein family known as connexins (Cxs) were identified as the cause of lymphatic dysfunction in some cases of inherited lymphedema. This dissertation explores the role of primarily two specific connexins, Cx37 and Cx43, and the transcription factor Foxc2 in the morphogenesis and function of the lymphatic vasculature in mice. To accomplish this, phenotypic characterization of mice with genetic deficiencies (knockout mice) in Cx37, Cx43, and/or Foxc2 was performed principally via necropsy, histological techniques (immuno-fluorescence microscopy and H&amp;E staining), and Evans blue dye (EBD) injections. Developmental abnormalities were found in lymphatic vascular growth, patterning, and remodeling in mice lacking Cx37, Cx43, Foxc2 or a combined deficiency of these proteins. Reductions or complete loss of lymphatic valves were a common finding in mice lacking one or more of these proteins. These valve deficits underlay lymphatic insufficiencies that resulted in lymphedema and chylothorax in some genotypes. Foxc2 was found to be a regulator of Cx37 expression. Moreover, Foxc2 was also dependent on Cx37 function for proper morphogenesis of lymph vessels. These findings pertaining to the expression of connexins in the lymphatic vasculature, their role in lymphatic valvulogenesis, and the interdependence of Cx37 and Foxc2 during lymph-vascular development represent my original contributions to human knowledge.</p>